Recent studies have shown that the extracellular calcium-sensing receptor (CaR) originally cloned from parathyroid (PT) plays a central role in setting the extracellular calcium concentration (Ca2+o). The CaR is expressed in PT cells and several kidney cells key playing roles in Ca2+o homeostasis. The essential, non redundant role of the CaR in calcium homeostasis has been documented by human diseases in which inactivating or activating CaR mutations produce hyper- or hypocalcemia, respectively, and the development of mice heterozygous or homozygous for targeted disruption of the CaR gene, which share many biochemical features of humans with one or two inactivated CaR alleles, respectively. It is not currently known, however, whether the CaR functions as a key, physiologically relevant extracelluar magnesium (Mg2+o)-sensing receptor, thereby controlling magnesium homeostasis the first specific aim to be addressed in this proposal. The availability of the cloned CaR has also made it feasible to test the effects of a wide variety of additional agents on receptor function. In preliminary studies for this proposal, two such factors, extracelluar pH and ionic strength, modulated the responsiveness of the CaR to Ca2+o and Mg2+o. Although large variations in these two parameters are unlikely to occur within the blood, both pH and ionic strength can vary in regions of the kidney expressing the CaR over ranges that have substantial effects on the receptor's function. Therefore, the second and third aims of this proposal are to document how changes in pH and ionic strength, individually or together, modulate the responsiveness of the CaR to Ca2+o and Mg2+o in physiologically relevant way. Finally, the lack of specific CaR antagonists has hindered attempts to determine which of the actions of Ca2+o and Mg2+o on a variety of cell types, including PT and renal cells, are actually mediated by the CaR. The final specific aim of the proposal, therefore, will be to characterize inhibitors of the CaR that have been identified in preliminary studies and utilize these inhibitors to investigate the role of the CaR in mediating the actions of Ca2+o and Mg2+o on carious aspects of PT and renal function. The results of these studies should document that in addition to sensing Ca2+o and Mg2+o, the CaR integrates key information about pH and ionic strength and also represents an important target for drug development of therapeutically useful CaR antagonists.